Pediococcus spp.-mediated competition interaction within Daqu microbiota determines the temperature formation and metabolic profiles.

Fermented microbiota is critical to the formation of microenvironment and metabolic profiles in spontaneous fermentation. Microorganisms generate a diverse array of metabolites concurrent with the release of heat energy. In the case of Daqu fermentation, the peak temperature exceeded 60°C, forming a typical high-temperature fermentation system known as high-temperature Daqu. However, microorganisms that cause the quality variation in Daqu and how they affect the functional microbiota and microenvironment in the fermentation process are not yet clear. This study adopted high-throughput sequencing and monitored the dynamic fluctuations of metabolites and environmental factors to identify the pivotal microorganism responsible for the alterations in interaction patterns of functional keystone taxa and quality decline in the fermentation system of different operational areas during the in situ fermentation process that had been mainly attributed to operational taxonomic unit (OTU)_22 (Pediococcus acidilactici). Additionally, we used isothermal microcalorimetry, plate inhibition experiments, and in vitro simulation fermentation experiments to explore the impact of Pediococcus spp. on heat generation, microorganisms, and metabolite profiles. Results showed the heat peak generated by Pediococcus spp. was significantly lower than that of Bacillus spp., filamentous fungi, and yeast. In addition, the preferential growth of P. acidilactici strain AA3 would obviously affect other strains to colonize through competition, and its metabolites made a significant impact on filamentous fungi. The addition of P. acidilactici strain AA3 in simulated fermentation would cause the loss of pyrazines and acids in metabolites. These evidences showed that the overgrowth of Pediococcus spp. greatly influenced the formation of high temperatures and compounds in solid-state fermentation systems. Our work illustrated the vital impact of interaction variability mediated by Pediococcus spp. for microbial assembly and metabolites, as well as in forming temperature. These results emphasized the functional role of Daqu microbiota in metabolites and heat production and the importance of cooperation in improving the fermentation quality.IMPORTANCEThe stable and high-quality saccharifying and fermenting starter in traditional solid-state fermentation was the prerequisite for liquor brewing. An imbalance of microbial homeostasis in fermentation can adversely impact production quality. Identification of such critical microorganisms and verifying their associations with other fermentation parameters pose a challenge in a traditional fermentation environment. To enhance the quality of spontaneous fermented products, strategies such as bioaugmentation or the control of harmful microorganisms would be employed. This work started with the differences in high-temperature Daqu metabolites to explore a series of functional microorganisms that could potentially contribute to product disparities, and found that the differences in interactions facilitated directly or indirectly by Pediococcus spp. seriously affected the development of microbial communities and metabolites, as well as the formation of the microenvironment. This study not only identified functional microbiota in Daqu that affected fermentation quality, but also demonstrated how microorganisms interact to affect the fermentation system, which would provide guidance for microbial supervision in the actual production process. Besides, the application of isothermal microcalorimetry in this study was helpful for us to understand the heat production capacity of microorganisms and their adaptability to the environment. This study presented a commendable framework for improving and controlling the quality of traditional fermentation and inspired further investigations in similar systems.

Identification and characterization of a novel phthalate-degrading hydrolase from a soil metagenomic library.

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 °C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.

Molecular cloning, expression and characterization of a novel feruloyl esterase from a soil metagenomic library with phthalate-degrading activity.

OBJECTIVES: To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. RESULTS: A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8 kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. CONCLUSION: The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

Daqu microbiota adaptability to altered temperature determines the formation of characteristic compounds.

Temperature plays a critical role in the performance of microbial communities during traditional solid-state fermentation. However, it remains unknown how temperature shapes microbiota, metabolism, and their relationship in Daqu fermentation. Here, we investigated the response of Daqu microbiota and metabolites to temperature by actual Daqu fermentation and simulated fermentation. First, volatile organic compounds were similar in both fermentation systems. Seventy-nine shared volatile compounds accounted for 94.5 %-96.5 % in Daqu fermentation and 66 %-95.6 % in the end of simulated fermentation, indicating that the formation of compounds in Daqu fermentation could be repeated effectively by simulated fermentation. The simulated fermentation showed the temperature gradient of 17 °C-60 °C significantly affected the formation and accumulation of volatile compounds. Aldehydes, acids, and pyrazines positively correlated with temperature (p < 0.05). Eight compounds were identified as characteristic compounds in high temperature (50-60 °C), including tetramethylpyrazine, trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 3-hydroxy-2-butanone, 2,3-dimethylpyrazine, benzaldehyde, acetic acid, and isovaleric acid. Next, we explored the force of temperature on microbial assembly and microbial interaction in simulated fermentation. Temperature significantly affected the composition of bacterial community (ANOISM, R = 0.779, P = 0.001) and fungi community (ANOISM, R = 0.664, P = 0.001). At the genus level, Weissella, Lactobacillus, Pediococcus, Saccharomycopsis Saccharomyces and Monascus dominated in 17-40 °C while Bacillus, Kroppenstedtia, Oceanobacillus, Lentibacillus, Rasamsonia, Thermoascus, Candida and Aspergillus were predominant genera in 50-60 °C. The succession of Bacillales, Lactobacillales, Eurotiales and Saccharomycetales adapted to changes in temperature. High temperature promoted microbial network complexity and a significant variation in microbial interactions. Furthermore, Procrustes analysis revealed a significant correlation between microbial community and volatile compounds (M2 = 0.6035, P < 0.001). Bacillus, Lentibacillus, Kroppenstedtia, and Oceanobacillus were significant contributors correlated to characteristic compounds. This study revealed the temperature-driven Daqu microbiota functioned as a critical contributor to promoting flavor formation and provided the theoretical basis for regulating fermentation in spontaneous fermentation systems.

Identification of a Novel Feruloyl Esterase by Functional Screening of a Soil Metagenomic Library.

A cosmid metagenomic library containing 1.3 × 105 clones was created from a soil sample. A novel gene (fae-xuan) encoding a feruloyl esterase was identified through functional screening. Primary sequence analysis showed that the gene consisted of 759 base pairs and encoded a protein of 252 amino acids. The gene was expressed in Escherichia coli BL21 (DE3) and the corresponding purified recombinant enzyme exhibited a molecular weight of 29 kDa. The FAE-Xuan showed high activity (40.0 U/mg) toward methyl ferulate with an optimal temperature and pH of 30 °C and 5.0, respectively. Besides methyl ferulate, FAE-Xuan can also hydrolyze methyl sinapate and methyl p-coumarate. The substrate utilization preferences and phylogenetic analysis indicated that FAE-Xuan belongs to type A FAE. FAE-Xuan was quite stable over a broad pH range from 3.0 to 10.0. The activity reduced remarkably in presence of Cu2+. FAE-Xuan can enhance the quantity of ferulic acid from de-starched wheat bran in presence of xylanase. The work presented here highlighted the effectiveness of metagenomic strategy in identifying novel FAEs with diverse properties for potential use in industrial production.

Author Correction: Identification of a Novel Feruloyl Esterase by Functional Screening of a Soil Metagenomic Library.

The original version of this article unfortunately contained a mistake in the image and caption of Fig. 6. The corrected version of the image and caption is shown here.

A Novel VIII Carboxylesterase with High Hydrolytic Activity Against Ampicillin from a Soil Metagenomic Library.

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50 °C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3 h at 60 °C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C ß-lactamases and family VIII esterases. As a consequence of its high sequence similarity with ß-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

Clone of plipastatin biosynthetic gene cluster by transformation-associated recombination technique and high efficient expression in model organism Bacillus subtilis.

Plipastatin, a cyclic lipopeptide, exhibits inhibitory activity against filamentous fungi and plays an important role in the prevention of plant diseases, and post-harvest preservation of fruits and vegetables. However, the application of plipastatin has been hampered by low yields in natural strains, while chemical synthesis is not feasible because of its complex chemical structure. In this study, a scarless genetic modification method was applied to construct a heterologous expression host (Bacillus subtilis 1A751 Δpps) by knocking out the natural plipastatin genes from B. subtilis strain 1A751. The core genes for plipastatin biosynthesis from B. amyloliquefaciens HYM12 were captured and assembled with sfp and degQ using transformation-associated recombination (TAR) cloning. The resultant gene cluster was introduced into B. subtilis 1A751 Δpps to generate strain B. subtilis 1A751 Δpps amyE::ppsA-E + sfp + degQ. Its fermentation products were analyzed and identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The results showed that the gene cluster for plipastatin synthesis was expressed successfully. This is the first time three gene fragments with significantly different DNA sizes (38.4, 0.3 and 0.8 kb) have been assembled by the TAR technique, thus we simultaneously established a method to express recombined large fragments in B. subtilis.